Bipolar cells of the retina are a highly diverse class of interneurons that receive direct input from photoreceptors, with 15 distinct types displaying unique gene expression patterns and functions in the visual circuitry. The comprehensive morphological and molecular information available, and powerful tools for genetic perturbation, make bipolar cells an ideal population for investigating how retinal interneurons become distinct during development. This project outlines a mentored (K99) phase during which the candidate can enhance existing expertise in genetic analysis of bipolar cell development through additional training, while applying these skills to address hypotheses emerging from new single-cell RNA-seq (scRNA-seq) datasets of mature and developing bipolar neurons. Specifically, this project is designed to interrogate how the combination of temporal differences in onset of proneural gene expression and extrinsic signaling mechanisms influence the decision of precursors of bipolar cells to become specific types. Aim 1 proposes to determine how the timing of proneural gene expression during postnatal development relates to the generation of distinct bipolar cell types, employing temporally controlled fate mapping of Neurog2-expressing cells and transient inhibition of proneural gene expression. In Aim 2, extrinsic cues regulating bipolar cell type identity during postnatal development will be investigated using a retinal explant system and pharmacological inhibitors of signaling pathway receptors identified as candidates from scRNA-seq expression datasets. A set of markers for all 15 bipolar cell types will be used for comprehensive phenotypic analysis of types. In Aim 3, enhancers of genes that are required for generating specific bipolar cell types, starting with Fezf2, will be analyzed for the binding of signal transduction pathway effectors and other transcriptional regulators. The K99 phase of the award will focus on parts of Aims 1-3, specifically temporally controlled fate mapping (Aim 1A), establishing the explant assay and applying inhibitors (Aim 2), and performing motif prediction and binding analysis for a known enhancer of Fezf2 (Aim 3), in addition to career development and training activities. In the R00 phase, the functional consequence of delaying proneural gene expression will be analyzed using transient Hes1 misexpression (Aim 1B), hits from the small-molecule inhibition experiment will be validated genetically and a temporal requirement for signaling will be further investigated (Aim 2), and enhancer analysis will be extended beyond Fezf2, to other genes required for generating particular bipolar cell types, including Isl1, Ebf1 and Prdm8 (Aim 3). The mentored phase will be conducted under the supervision of Dr. Constance Cepko, with additional advisory contributions from Dr. Paola Arlotta (Harvard) and faculty in the Genetics Department at Harvard Medical School. The extensive resources and career development opportunities available at Harvard Medical School, Dr. Cepko's mentorship, and the research activities planned in the K99 period will enable the candidate to achieve the long- term goal of becoming an independent investigator dedicated to the study of retinal neurogenesis.